1. Field of the Invention
The present invention relates to novel and useful (1) intramolecularly cross-linked, stromal-free deoxyhemoglobin, and (2) intramolecularly cross-linked, stromal-free oxyhemoglobin. The intramolecularly cross-linked stromal free hemoglobins of this invention have the property of reversibly binding gaseous ligands such as oxygen, and they are useful as blood substitutes for transporting and supplying oxygen to vital tissues and organs, as blood plasma substitutes as sources of oxygen for biological systems, and for furnishing oxygen to oxygen-consuming environments.
2. Description of the Prior Art
Hemoglobin is present in the blood of mammals and it has the fundamental property in solution of reversible oxygenation. In its natural form, mammalian hemoglobin is a conjugated, non-crosslinked protein having a molecular weight of 64,500 and structurally comprised of two pairs of sub-units. Each sub-unit contains a heme group and a polypeptide chain, called globin. In mammals, hemoglobin is present in erythrocytes along with stroma which consists of proteins, phospholipids and cholesterol. Clinical Hematology, by Wintrobe, 6th Ed., pages 138 to 199, 1967, published by Lea and Febiger, Philadelphia, Pa. The reaction of isolated, bovine hemoglobin containing stroma with glutaraldehyde is known to the art in Histochemical J., Vol. 2, pages 137 to 150, 1970, wherein Hopwood used an excess of glutaraldehyde to obtain an insoluble precipitate. Similarly, the reaction of whole blood proteins with glutaraldehyde leading to a water insoluble glue is disclosed by Karjala, et al, in the U.S. Pat. No. 3,294,564. The interaction of the collagen and collagen degradation product gelatin with diisocyanates and other polycoupling agents, including aldehydes, is reported by Campbell in U.S. Pat. No. 2,591,133, by Linder, et al, in U.S. Pat. No. 3,057,782, and by Bowes in Biochimica et Biophysica Acta, Vol. 168, pages 341 to 352, 1968. The carboxyalkylation of globin for use as a plasma expander is taught by Biddison in U.S. Pat. No. 2,719,837; however, the products obtained by the above reactions lacked the ability to transport oxygen and in consequence thereof, they did not enter into general use. In U.S. Pat. No. 2,527,210, Bower disclosed the use of hemoglobin for treating wounds, in U.S. Pat. Nos. 3,000,836 and 3,519,572, Ginsburg and Kita disclosed blood preparations having utility as standards for measuring hemoglobin. In Federation Proceedings, Vol. 34, pages 1458 to 1460, 1975, Mok et al. disclosed the reaction of hemoglobin solutions containing stromal-free protein with diimidate esters leading to intramolecularly cross-linked and intermolecular complexes. In U.S. Pat. No. 3,925,344, Mazur disclosed a plasma protein substitute consisting of intramolecularly cross-linked hemoglobin formed by using diimidate cross-linking agents. In these latter two references, the reaction was carried out apparently in an air environment and free of reducing conditions which leads to hemoglobin in the oxygenated form. A diimidated cross-linked hemoglobin similar to the product of U.S. Pat. No. 3,925,344 also is disclosed in Netherland Pat. No. 7,041,140.